Abstract. Genomic instability is a major contributing factor to the development and onset of age-related diseases such as cancer. Aberrant regulation of the spatial-temporal timing of DNA replication is a critical factor involved in genomic instability and increased cancer risk. Chromatin structure plays a fundamental role in coordinating the timing of DNA replication during G1 and S phases of cell cycle. Chromatin is a highly ordered structure that contains DNA, histones, and other chromosomal proteins. Posttranslational modifications of histones influence the outcomes of numerous biological processes including transcription, replication, and chromosome maintenance. Histone 3 lysine 9 tri-methylation (H3K9me3) is considered a hallmark of heterochromatin, a repressive structure found at repetitive and telomeric regions in the genome. Alterations in H3K9me3 and heterochromatin levels result in aberrant replication timing and the onset of genomic instability and cancer. Understanding how the dynamics of methylation and heterochromatin formation and maintenance are regulated throughout cell cycle is critical for uncovering both basic principals governing replication timing, but most importantly, identifying regulatory factors that could contribute to replication timing defects and genomic instability observed in cancer. We have recently identified the JMJD2A H3K9/36 tri-demethylase as a regulator of chromatin accessibility, heterochromatin (HP13), DNA replication timing and S phase progression. We also demonstrated that JMJD2A levels and localization are regulated over cell cycle by ubiquitination, which directly impacts the role of JMJD2A in S phase progression. These data are exciting since regulating ubiquitination has emerged as a therapeutic strategy in cancer treatment. We hypothesize that HP13 and the ubiquitination of JMJD2A regulate the targeting of specific genomic regions so that orderly cell cycle progression occurs. This grant proposal will determine the impact ubiquitination has on JMJD2A cell cycle function (Aim 1) and determine how JMJD2A alters chromatin structure so that DNA replication timing is properly spatially and temporally regulated (Aim 2). In aim 1, we will use proteomic and biochemical approaches to determine the sites/regions that are modified within JMJD2A, the enzymes involved in adding and removing the ubiquitin, and their impact on JMD2A- dependent S phase progression. In aim 2, we will use genomics to determine the regions regulated by both JMJD2A and heterochromatin throughout cell cycle as well as determine the impact JMJD2A has on replication timing (i.e., initiation and/or elongation). These studies will significantly impact our basic understanding of S phase progression and clinical understanding of JMJD2A overexpressing tumors.